Sometimes help comes from unexpected places. The threat of climate change, rising fuel costs, and dwindling supplies of petroleum have a lot of people looking for ways around these serious problems. And “pond scum” just might be the solution—or, more appropriately, blue green algae, photosynthetic microbes.
Blue green algae, or cyanobacteria, are ideally suited as producers of biofuels for a number of reasons:
- these microbes efficiently convert solar energy into fuel compounds by recycling carbon dioxide form the atmosphere;
- they do not need a carbon feed to make biofuels;
- they generate biofuels without competing with the agricultural industry for arable land;
- they are easy to engineer genetically.
However, the fatty acid products that serve as raw materials for biofuels must be extracted from the cells. This presents a major drawback to using cyanobacteria to produce biofuel. The extraction process is energy-intensive, requiring scientists to harvest the cells from the growth medium, dry them, and then extract the fatty acids with a variety of solvents.
Recently, synthetic biologists and biotechnologists from Arizona State University (ASU) discovered a solution. They recognized that the extraction hurdle can be overcome if the cyanobacteria are redesigned to overproduce fatty acids1. They could then secrete the fatty acids into the growth medium. In this way the fatty acids can be readily isolated from the growth medium, thereby averting many of the steps required to extract biofuels from the cyanobacterial cells.
To reengineer the cyanobacterium Synechocystis sp. PCC 6803, the ASU researchers focused on the acyl carrier protein (ACP). This protein forms a complex with fatty acids and represents the end product of fatty acid biosynthesis. The complex forms when the cell’s machinery joins a fatty acid to the protein via a special type of chemical linkage called a thioester bond. The accumulation of fatty acid-ACP inside the cell inhibits the metabolic pathways that produce fatty acids. This feedback inhibition serves as a regulatory device that keeps the cell from overproducing fatty acids.
The researchers looked for ways to inhibit the production of the fatty acid-ACP complex and to break it down once formed. To do this they made use of an enzyme from E. coli. This microbe produces a protein called ACP thioesterase I. Other scientists learned that mutant forms of ACP thioesterase I will cleave the thioester bond joining the fatty acid to the ACP complex. (Interestingly, biochemists have noted that the mutated form of the thioesterase also promotes secretion of fatty acids into the growth medium.)
To cause Synechocystis to overproduce and secrete fatty acids, the ASU scientists incorporated the altered form of E. coli ACP thioesterase I into the cyanobacterium’s genome. They also engineered the E. coli gene to regulate it in one of two ways: (1) one strain continuously produces ACP thioesterase I; and (2) the other produces this enzyme only when nickel ions are present in the growth medium. Such regulations allowed the researchers to turn the E. coli gene on and off.
The next step in the reengineering process involved disabling via genetic manipulations the gene that codes for the enzyme acyl-ACP synthetase. This enzyme attaches fatty acids to the ACP protein. By disrupting the gene, the researchers impaired Synechocystis’sability to make fatty acid ACP protein complexes, thus, preventing the inhibition of fatty acid biosynthesis.
In order to increase Synechocystis’s fatty acid production, the researchers manipulated this microbe’s genome so that it produced unusually high levels of acetyl-CoA carboxylase, the enzyme that catalyzes the first step of the metabolic pathway that makes fatty acids. They also genetically altered the production of a gene that produces a protein phycocyanin, which plays a role in harvesting energy from sunlight for carbon fixation. Along these lines, the scientists also increased the production of the protein ribulose 1,5-bisphosphate carboxylase, the enzyme that catalyzes the first reaction of the carbon fixation cycle. They also shut down the activity of two genes responsible for encoding proteins that funnel the end products of carbon fixation away from fatty acid biosynthesis.
During the course of the genetic engineering work, the team of synthetic biologists learned that the thickness of the cell wall modulates the amount of fatty acid secreted by the cells. This discovery led them to alter the production of a protein that helps form the S-layer of the cell envelope.
The net effect of all this work was the production of a strain of Synechocystis that grew rapidly during the cell culture’s stationary phase and proved capable of producing over 150 mg of fatty acids per liter of culture per day.
The Production of Biodiesel and the Case for Intelligent Design
As I pointed out in two previous articles on reengineering E. coli for biodiesel production, the ingenuity and thoughtful planning of the researchers (based on decades of accumulated knowledge and insight) made it possible for them to engineer the cyanobacterium Synechocystis sp. PCC 6803 to make biodiesel.
It’s worth noting that, as marvelous as this achievement is, the researchers didn’t create this metabolic capability from scratch. They pieced together the pathway using modified enzymes taken from a variety of sources and by manipulating the activity of endogenous Synechocystis genes.
In the end, it’s fair to say that this novel metabolic process was intelligently designed. In fact, biochemists describe this type of work as rational design. The amount of effort invested in reengineering existing metabolic systems to make biodiesel raises provocative questions. Is it reasonable to maintain that life’s chemistry originated and evolved through undirected processes? Doesn’t this work provide direct, empirical evidence that biochemical systems require the work of a rational agency in order to come into existence and to undergo significant change?
Even though many argue work in synthetic biology supports the evolutionary paradigm, the case for intelligent design gets help from unexpected places—the attempts to reengineer non-natural life-forms.